PROJECT SUMMARY Down syndrome (DS), trisomy 21, is the most common intellectual disorder affecting millions and also is a form of early-onset Alzheimer Disease (AD). Understanding of DS pathology has been hindered by the complexity caused by overexpression of the ~250 genes on chromosome 21 (chr21). Many reports describe a diverse range of developmental and functional pathologies in the DS brain. However, there is conflicting evidence even as to which brain regions or cell types are impacted. Furthermore, few studies address when pathology arises and when it remains reversible. Therefore, better understanding of the exact impact of DS on brain development and function, when said effects occur, and identification of mechanistic causes in specific brain cell types is critical. Reports from mouse models, human induced pluripotent stem cells (iPSCs), and post-mortem DS brain samples regarding the impact of DS on brain development and function are conflicted. Furthermore, when during development the impacts of DS occur and which chr21 genes are responsible are unknown. Preliminary results show that the clonal variability between isogenic disomic and trisomic cell lines was too great to establish any neurodevelopmental effect of trisomy even when organoids variability was well controlled. Surprisingly, AD- related Aβ pathology was still observed. To circumvent variability between cell lines our strategy will be to manipulate over-expression of chr21 in several trisomic cell lines. By inserting an inducible XIST transgene into the extra chr21, our lab has demonstrated comprehensive chromosomal silencing using endogenous machinery for dosage compensation. Our XIST-inducible system for “trisomy silencing” reduces variability by allowing the study of nearly identical cells, with or without extra chr21 expression. Using this system, the Lawrence lab has demonstrated that DS potentially delays neurogenesis by prolonging neural progenitor cell (NPC) fate during neuronal monolayer differentiation. Prolonged NPC cycling is known to alter cell fate and therefore we will examine later stages of neurodevelopment that may alter brain cell-type composition. At the same time, we will examine if trisomy silencing can correct an established AD-related cell pathology in DS organoids. We will also use these approaches to examine the impact of trisomy 21 on global transcription in specific cell types relating to cell pathologies. A current theme in DS research is that trisomy 21 causes very broad changes in expression of non-chr21 genes. Through transcriptomics many studies have reported that genome-wide impacts may alter specific genes/pathways, impacting both the development and activity of different neural cell types. However, work in our lab suggests other differences between samples, not due to trisomy, may account for much of this “global perturbation”. The hypothesis for this proposal is that trisomy 21 alters the relative proportions of neural cell types in the br...